9-Deoxy-9,10-epoxide-daunomycinone

ABSTRACT

Compounds having the formula: ##STR1## wherein (a) when 
     R 1  is --COCH 3  or --COCH 2  OH, 
     R 2  is --OH, R 3  is --OCH 3  and R 4  is --H; 
     (b) when 
     R 2  is --COCH 3  or --COCH 2  OH; 
     R 1  is --OH, R 3  is --H and R 4  is --OCH 3  ; 
     and which are useful in treating certain mammalian tumors, are prepared from 9,10-anhydro-N-trifluoroacetyl daunorubicin, a known compound. The invention described herein was made in the course of work under a grant from the United States Department of Health, Education and Welfare.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 941,847, filed Sept. 13,1978.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new antitumor glycosides of theanthracycline series, novel intermediates used for making them,processes for the preparation of said glycosides and the use thereof.

2. The Prior Art

Daunorubicin (also known as daunomycin) and doxorubicin (also known asadriamycin), of which the present compounds are derivatives, are knownand are known to be useful in treating certain mammalian tumors.

The compound 9,10-anhydro-N-trifluoroacetyldaunorubicin (X), which isthe starting material for the compounds of the present invention is aknown compound which is described in British Patent Specification No.53456/76, owned by the unrecorded assignee hereof.

SUMMARY OF THE INVENTION

The present invention provides, in one aspect thereof, a new class ofantitumor anthracyclines of the formula: ##STR2## I: R¹ =COCH₃ ; R² =OH;R³ =OCH₃ ; R⁴ =H; X=COCF₃ ;

II: R¹ =COCH₃ ; R² =OH; R³ =OCH₃ ; R⁴ =H; X=H;

III: R¹ =COCH₂ OH; R² =OH; R³ =OCH₃ ; R⁴ =H; X=H;

IV: R¹ =OH; R² =COCH₃ ; R³ =H; R⁴ =OCH₃ ; X=COCF₃ ;

V: R¹ =OH; R² =COCH₃ ; R³ =H; R⁴ =OCH₃ ; X=H;

VI: R¹ =OH; R² =COCH₂ OH; R³ =H; R⁴ =OCH₃ ; X=H.

Among these six compounds, the four (II, III, V and VI) wherein X is Hare the most important.

The invention also provides, in another aspect, a novel method forpreparing these compounds utilizing several novel intermediates, whichare also within the scope of the invention.

In yet another aspect, the invention provides pharmaceuticalcompositions which include the novel antitumor compounds of theinvention.

Finally, the invention also provides a method of using the novelantitumor compounds of the invention in the treatment of certainmammalian tumors.

The preparation of the compounds of formulae I-VI is based on thesynthesis of the tetracyclic aglycones of the formula: ##STR3## VII: R¹=OH; R² =COCH₃ ; R³ =OCH₃ ; R⁴ =H; VIII: R¹ =COCH₃ ; R² =OH; R³ =H; R⁴=OCH₃ ;

and on the subsequent condensation of the aglycones VII and VIII withthe known halosugar, 1-chloro-N,O-trifluoroacetyldaunosamine (IX):##STR4## to form the corresponding N,O protected glycosides, which aftertreatment with methanol to eliminate the O-protecting trifluoroacetylgroup, form the corresponding N-protected glycosides I and IV. Afterhydrolysis of the N-trifluoroacetyl-protecting group on the sugarmoiety, 10-methoxydaunorubicin (II) and9,10-diepi-10-methoxydaunorubicin (V) are obtained. The correspondingdoxorubicin analogs (III) and (VI) are prepared from (II) and (V),repectively, via the 14-bromo derivatives, in accordance with the methoddescribed in U.S. Pat. No. 3,803,124 which is owned by the unrecordedassignee hereof. The starting materials for the preparation of the newglycosides (I-VI) of the invention therefore, are the anthracyclinones(VII) and (VIII) which were previously unknown. The novelanthracyclinones (VII) and (VIII) are synthesized, starting from9,10-anhydro-N-trifluoroacetyldaunorubicin (X), which is described inBritish Patent specification No. 53456/76 (owned by the unrecordedassignee hereof). The synthetic reaction scheme is set forth below: Inorder to effect epoxidation of the conjugated double bond present at theC-9, C-10 position in (X), it is first necessary to reduce the ketofunction to the corresponding α,β-unsaturated alcohol (XI). Thisreduction is effected using sodium cyanoborohydride in a suitablewater-miscible-organic solvent, such as dioxane or dimethoxyethane, inthe presence of a mineral acid, and yields quantitatively thecorresponding 13-dihydroderivative (XI). Compound (XI) is then subjectedto an expoxidation reaction using m-chloroperbenzoic acid in an aproticsolvent, such as methylene chloride, chloroform or acetone. Theepoxidation reaction proceeds at a temperature between 25° and 80° C. togive 9,10-epoxide-13-dihydro-N-trifluoroacetyldaunorubicin (XII), as anepimeric mixture. The regeneration of the keto function, with thecontemporaneous cleavage of the glycosidic linkage, is performed byoxidation with dimethyl sulfoxide and dicyclohexylcarbodiimide, usingpyridinium trifluoroacetate as a catalyst. The course of this oxidationreaction is influenced by the amount of catalyst; using the ratio ofsubstrate to salt of 1:1, compound (XIII) is obtained in high yield. Thesubsequent introduction of the methoxy group, which is performed byopening of the oxirane ring of compound (XIII) with methanol, in thepresence of a catalytic amount of p-toluensulfonic acid, gives, in theapproximate ratio of 7:2, a mixture of the aglycones (VII) and (VIII)which are separated by chromatography on silica gel. Compounds (VII) and(VIII) differ stereochemically at the C-9 and C-10 centers. This isdemonstrated by the fact that only compound (VII) forms a7,9-isopropylidenderivative (XIV) by treatment with2,2-dimethoxypropane, which shows that the hydroxyl groups at C-7 andC-9 are cis. The pmr spectra of (VII) and (VIII) show that the C-10 Hhas an equatorial orientation in (VII) and an axial orientation in(VIII). Treatment of (VIII) with 2,2-dimethoxypropane gives7-methoxy-9,10-diepi-10-methoxydaunomycinone (XV).

The coupling reaction between the aglycones (VII) and (VIII) and the N,Oprotected halosugar (IX) to form the glycosidic linkage, is carried outin a suitable organic solvent, such as chloroform, methylene chloride ortetrahydrofuran in the presence of a silver salt as catalyst. Thethereby obtained N,O protected glycosides are first treated withmethanol to eliminate the O-protecting trifluoroacetyl group on thesugar moiety, to give the N-protected glycosides (I) and (IV). Thesecompounds, upon mild alkaline treatment, are converted in quantitativeyield to 10-methoxydaunorubicin (II) and9,10-diepi-10-methoxydaunorubicin (V), respectively.

The corresponding doxorubicin analogs (III) and (VI) are respectivelyobtained from (II) and (V) via the 14-bromo derivatives, according tothe procedure described in U.S. Pat. No. 3,803,124. The new compounds(I-VI) display antimitotic activity and are therefore useful therapeuticagents for the treatment of tumor diseases in mammals.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are given to better illustrate the inventionwithout, however, being a limitation thereof.

EXAMPLE 1 9,10-Anhydro-13-dihydro-N-trifluoroacetyldaunorubicin (XI)

6.0 Grams (10 mmoles) of 9,10-anhydro-N-trifluoroacetyl daunorubicin (X)were dissolved in 2000 ml. of methanol. The solution was acidified with50 ml. of 0.1 N aqueous hydrochloric acid and then reacted with anaqueous solution of NaCNBH₃ (4.0 g. in 200 ml. of H₂ O). The reactionmixture was stirred at room temperature for 48 hours, while keeping thepH below 4 by the addition of 0.1 N aqueous hydrochloric acid. Afterneutralization with an excess of solid NaHCO₃, the solution wasevaporated to a residue under vacuum and the residue, after beingdissolved in chloroform, was washed with water. The chloroform solutionwas dried over anhydrous Na₂ SO₄ and the solvent finally removed undervacuum to yield crude9,10-anhydro-13-dihydro-N-trifluoroacetyldaunorubicin (XI). Pure9,10-anhydro-13-dihydro-N-trifluoroacetyldaunorubicin (XI) was obtainedby chromatographic purification on a column of silicic acid using as theeluting agent the system CHCl₃ :(CH₃)₂ CO (95:5 v/v). The pure compoundmelts at 165° C. (dec.). Visible spectrum (CHCl₃) maxima at 520, 556 nm.

EXAMPLE 2 9-Deoxy-9,10 -epoxide-13-dihydro-N-trifluoroacetyldaunorubicin(XII)

To a solution of 8 g. (3.28 mmoles) of9,10-anhydro-13-dihydro-N-trifluoroacetyldaunorubicin (XI) in 400 ml. ofchloroform, there were added 1.08 g.; 6 mmoles of m-chloroperbenzoicacid, and the reaction mixture was warmed at 80° C. for 3 hours. Theinitial cherry color of the solution gradually changed to red. Thereaction solution was then cooled and washed with an aqueous saturatedsolution of NaHCO₃, and then with water, after which it was finallydried over anhydrous Na₂ SO₄. The solvent was evaporated to a residueunder vacuum. The residue (2.0 g.), which exhibited, in the visiblespectrum (CHCl₃), maxima at 490, 504 and 540 mμ is in agreement withwhat would be expected as a result of the disappearance of the doublebond at the C-9, C-10 position of (XI), was a mixture of epimericepoxides and was used without further purification in the followingexample.

EXAMPLE 3 9-Deoxy-9,10-epoxide-daunomycinone (XIII)

To a stirred solution of 3.85 g. (6 mmoles) of9-deoxy-9,10-epoxide-13-dihydro-N-trifluoroacetyldaunomycin (XII) undera nitrogen atmosphere, in 100 ml. of anhydrous dimethylsulfoxide, therewere added, one after the other, 3.8 g. (18 mmoles) ofdicyclohexylcarbodiimide, 0.5 ml. (6 mmoles) of anhydrous pyridine and0.23 ml. (3 mmoles) of trifluoroacetic acid. The resulting mixture wasstirred at room temperature for 15 hours and then diluted with 500 ml.of chloroform. The chloroform solution was thoroughly washed with water,dried and evaporated to a residue. The residue was taken up in ethylacetate, the insoluble dicyclohexylurea was filtered off and thefiltered solution evaporated to a residue to give9-deoxy-9,10-epoxide-daunomycinone (XIII) in quantitative yield.

IR: 1720 cm.sub.ν⁻¹ _(C)═O ; 1580 and 1620 cm.sub.ν⁻¹ _(C)═O quinone.

NMR (CDCl₃): at 2.27 (1, CH₃ --C═O); 4.10 (s, OCH₃) and 4.18δ (1, H-10).

EXAMPLE 4 10-Methoxy-daunomycinone (VII) and9,10-diepi-10-methoxydaunomycinone (VIII)

A solution of 4.3 g. of 9-deoxy-9,10-epoxidedaunomycinone (XIII) in 500ml. of anhydrous methanol was warmed at reflux temperature for 15 hoursin the presence of a catalytic amount of p-toluensulfonic acid. Thereaction mixture was then cooled and evaporated to a residue which wasthen dissolved in 300 ml. of chloroform, washed with an aqueous 5%solution of NaHCO₃, water, dried over anhydrous Na₂ SO₄ and againevaporated to a residue. The thusly obtained crude material was amixture of compounds (VII) and (VIII) in the approximate ratio of 7:2.The mixture was chromatographed on a column of silicic acid using themixture ethylacetate-toluene-petroleum ether (3:2:2 v/v) as the elutingagent. Pure 10-methoxydaunomycinone (VII); (1.5 g.) and9,10-diepi-10-methoxydaunomycinone (VIII); (0.42 g.), 72δ overall yield,were obtained. 10-Methoxydaunomycinone (VII): m.p. 220° C. (dec.);[α]_(D) ²⁰ =+206 (c=0.1, CHCl₃);

MS: m/e 428 (M+): 396 (M--CH₃ OH), 353 (M--CH₃ OH--CH₃ CO).

NMR (CDCl₃): 3.51 (s, C-10-OCH₃); 4.66 (d, C-10 H); 5.31 (q, C-7H), 13.6and 14.07δ (s, OH phenolic).

9,10-Diepi-10-methoxydaunomycinone (VIII): m.p. 156° C. (dec.):

MS: m/e (428 (M+).

NMR (CDCl₃): 3.64 (s, C-10-OCH₃); 4.89 (s, C-10H), 5.12 (q, C-7H), 13.80and 14.21δ (s, OH phenolic).

EXAMPLE 5 7,9-Isopropyliden-10-methoxydaunomycinone (XIV)

To a solution of 0.1 g. of 10-methoxydaunomycinone (VII) in 10 ml. ofanhydrous dioxane there were added 5 ml. of 2,2-dimethoxypropane and acatalytic amount of p-toluensulfonic acid. The reaction mixture was keptat 50° for 48 hours and then diluted with 50 ml. of chloroform. Thethusly diluted solution was washed with an aqueous saturated solution ofNaHCO₃, water and then dried over anhydrous Na₂ SO₄. The crude residue,obtained by evaporation of the organic solvent, was chromatographed on acolumn of silicic acid using the mixture chloroform-actone (95:5 v/v) aseluting agent. Pure 7,9-isopropyliden-10-methoxydaunomycinone (XIV) wasobtained.

MS: m/e 468 (M⁺); 410 (M-(CH₃)₂ CO); 378 (M-(CH₃)₂ CO-CH₃ OH).

NMR (CDCl₃): 1.2 and 2.47 (s, 2CH₃), 5.47δ )m, C-7H).

EXAMPLE 6 7-Methoxy-9,10-diepi-10-methoxydaunomycinone (XV)

Treatment of 9,10-diepi-10-methoxydaunomycinone (VIII) with2,2-dimethoxypropane, as described in Example 5, afforded7-methoxy-9,10-diepi-10-methoxydaunomycinone (XV).

MS: m/e 442 (M⁺).

EXAMPLE 7 10-Methoxydaunorubicin hydrochloride (II)

To a solution of 0.43 g. (1 mmole) of 10-methoxydaunomycinone (VII) in200 ml. of anhydrous methylene chloride were added 0.43 g. (1.2 mmoles)of 1-chloro-N,O-trifluoroacetyldaunosamine (IX). Then 0.32 g. (1.2mmoles) of AgSO₃ CF₃, dissolved in 26 ml. of anhydrous ether was addedto the solution at room temperature with vigorous stirring over a periodof 10 minutes. Finally, 0.2 ml. (1.4 mmoles) of anhydrous collidine wasadded to the reaction mixture. After 40 minutes, the mixture was treatedwith a saturated aqueous solution of NaHCO₃ and the separated organicphase was evaporated under vacuum. The resulting residue was dissolvedin 100 ml. of methanol and kept at room temperature for 5 hours. Theresidue, which resulted from the removal of the solvent, waschromatographed on a column of silicic acid using the mixturechloroform-acetone (4:1 v/v) as the eluting agent. In addition tounreacted 10-methoxydaunomycinone (VII), there was also obtained 0.26 g.of pure 10-methoxy-N-trifluoroacetyldaunorubicin (I); m.p. 190° C.(dec.): TLC on Kieselgel plate F₂₅₄ (Merck) using the solvent systemCHCl₃ -(CH₃)₂ CO (4:1 v/v): Rf 0.3;

NMR (CDCl₃): 1.30 (d, CH₃ -CH); 3.52 (s, C-10 OCH₃); 5.30 (m, C-7H) and5.53δ (m, C-1'-H×WH=7HZ).

The compound (I); 0.26 g. was dissolved in 50 ml. of 0.1 N aqueoussodium hydroxide and after 30 minutes at 0° C., the solution wasadjusted to pH 8.6 and repeatedly extracted with chloroform. Thecombined chloroform extracts, after being dried over anhydrous Na₂ SO₄,were concentrated to a small volume and acidified at pH 4.5 with 0.1 Nmethanolic hydrogen chloride to allow crystallization of10-methoxydaunerubicin (II), as the hydrochloride; m.p. 159° C. (dec.);[α]_(D) ²⁰° +316° (c 0.05, CH₃ OH); TLC on Merck Kieselgel HF₂₅₄ plateusing solvent system CHCl₃ -CH₃ OH-H₂ O (13:6:1 v/v): Rf 0.37.

EXAMPLE 8 9,10-Diepi-10-methoxydaunorubicin (V)

The coupling reaction between 9,10-diepi-10-daunomycinone (VIII) and thehalosugar, i.e., 1-chloro-N,O-trifluoroacetyldaunosamine (IX), asdescribed in Example 7, yielded9,10-diepi-10-methoxy-N-trifluoroacetyldaunorubicin (IV), which, after amild alkaline treatment with 0.1 N aqueous sodium hydroxide, for 30minutes at 0° C., gave 9,10-diepi-10-methoxydaunorubicin, isolated asthe hydrochloride (V), m.p. 140° (dec.); [α]_(D) ²⁰° +252 (C 0.05,MeOH).

EXAMPLE 9 10-Methoxydoxorubicin (III)

A solution of 10-methoxydaunorubicin (II) in a mixture of methanol anddioxane was treated with bromine to give the corresponding14-bromoderivative which was subsequently treated with an aqueoussolution of sodium formate at room temperature for 100 hours accordingto the technique disclosed in U.S. Pat. No. 3,803,124 to obtain10-methoxydoxorubicin (III), isolated as the hydrochloride m.p. 195°(dec.) TLC on Merek Kieselgel HF₂₅₄ plate using solvent system CHCl₃-CH₃ OH-H₂ O AcOH (8:2:0.6:1.4 v/v) Rf 0.45.

EXAMPLE 10 9,10-Diepi-10-methoxydoxorubicin (VI)

As in Example 9, by following the technique disclosed in U.S. Pat. No.3,803,124, the treatment of 9,10-diepi-10-methoxydaunorubicin (V) withbromine and then with sodium formate, yielded9,10-diepi-10-methoxydoxorubicin (VI) which was isolated as thehydrochloride.

BIOLOGICAL ACTIVITY

The compounds according to the invention were tested under the auspicesof NCl-National Institute of Health, Bethesda, Maryland, againstlymphocytic leukemia P₃₈₈ according to the procedure described in CancerChemotherapy Reports, part 3, vol. 3, page 9 (1972). The data reportedin the table below show the antitumor activity of the new anthracyclinederivatives.

                  TABLE                                                           ______________________________________                                        Antitumor activity of 10(R)-methoxydaunorubicin and 10(R)-                    methoxydoxorubicin as compared with daunorubicin and                          doxorubicin                                                                                       Dose                                                      Compound            (mg./kg.)  T/C %                                          ______________________________________                                        Daunorubicin        16          90                                                                8           98                                                                4          119                                                                2          124                                            10(R)-Methoxydaunorubiein                                                                         12.5       133                                                                6.25       115                                                                3.13       110                                            Doxorubicin         16         108                                                                8          171                                                                4          133                                                                2          129                                                                1          119                                            10(R)-Methoxydoxorubicin                                                                          50         104                                                                25         115                                                                12.5       127                                                                6.25       125                                                                3.13       108                                            9-epi-10(S)-Methoxydauno-                                                                         50         126                                            rubicin             25         132                                                                12.5       118                                                                6.25       102                                            ______________________________________                                    

The new compounds were tested in vivo on CDF mice infected with tumorcells. The i.p. injections were made on days 5, 9 and 13 (4 daysinterval between each injection) starting from fifth day after tumortransplantation in the mice. The median survival time expressed as percent of controls (T/C %) are reported.

Variations and modifications can, of course, be made without departingfrom the spirit and scope of the invention.

Having thus described our invention, what we desire to secure by LettersPatent and hereby claim is:
 1. The compound having the formula: ##STR5##